The neuronal ceroid lipofuscinoses or Batten disease (BD) is one of a broad class of severe neurodegenerative diseases that are known as lysosomal storage disorders (LSDs). BD is characterized by the intracellular accumulation of storage material in neural tissues and progressive loss of neurological functions and vision that primarily affect children. CLN2 is a specific type of BD that results from mutations in the TPP1 gene causing an insufficiency or complete lack of a soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Last year, the first FDA-approved treatment, intraventricular infusions of a recombinant form of human TPP1, was shown to provide symptomatic improvements in pediatric patients. Unfortunately, this invasive procedure carries a high risk of adverse effects. The less invasive systemic administration provides no therapeutic benefits, because the blood brain barrier (BBB) severely restricts transport of macromolecules - including TPP1- to the brain. To circumvent this problem, we propose using extracellular vesicles (EVs) released by macrophages as biocompatible nanocarriers for systemic delivery of TPP1. Comprised of cellular membranes with multiple adhesive proteins on their surface, EVs are known to specialize in cell-cell communications facilitating transport of proteins and genetic material to target cells. Our research has previously demonstrated that therapeutic proteins, including TPP1, can be efficiently incorporated into EVs without losing their biological activity. In particular, TPP1 was loaded into EVs using two methods: (i) transfection of parental EV-producing macrophages with TPP1-encoding plasmid DNA (pDNA), or (ii) loading therapeutic protein TPP1 into naive empty EVs. The resulting EVs carrier ensemble was shown to readily migrate into the brains of late-infantile neuronal ceroid lipofuscinosis (LINCL) mice upon systemic administration. Importantly, multiple lines of evidence for therapeutic efficacy were observed in LINCL mice, including significant neuroprotection and improved life span. Noteworthy, we developed different methods for EVs isolation, purification, characterization, and storage in sufficient quantities for therapeutic application. In the current proposal, we will utilize two EV-based formulations of TPP1 to demonstrate the proof of concept using a BD mouse model, LINCL mice. Planned studies include: (SA1) elucidation the nature of selective fingerprinting of macrophage-derived EVs, and mechanism of EVs interactions and TPP1 facilitated uptake in cells of neurovascular unit; (SA2) evaluation of brain bioavailability for EV-TPP1 with MRI and optical imaging in vivo, and (SA3) validation the therapeutic potential of this novel drug delivery system by measuring its neuroprotective and anti-inflammatory effects in LINCL mice. The proposed research addresses a critical problem in the effective delivery of therapeutic proteins to the central nervous system (CNS), and will provide fundamental insights into, how EVs communicate with target brain cells, and selectively deliver their cargo.